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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P13/00—Preparation of nitrogen-containing organic compounds
  • C12P13/04—Alpha- or beta- amino acids
  • C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P13/00—Preparation of nitrogen-containing organic compounds
  • C12P13/04—Alpha- or beta- amino acids
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms

Definitions

• the invention relates to a method for the selection (screening) of productive strains of microorganisms which secrete amino acids for use in fed-batch fermentation.
• L-lysine The essential amino acid L-lysine is of great industrial importance as a food and animal feed additive and as an active ingredient and component of pharmaceutical products.
• Fed batch fermentation is the most important process for the production of L-lysine.
• Coryneform bacteria of the genera Corynebacterium and Brevibacterium in particular are used in production. Mutations have changed the regulation of the lysine biosynthesis of these strains in such a way that they produce lysine beyond their own requirements and excrete into the medium.
• overproducers can be obtained by searching for mutants in which individual steps of the amino acid metabolism are blocked (e.g. hese or thr auxotrophs), which are resistant to one or more analogs of lysine or which contain further mutations.
• High-performance strains have several Auxotrophies, analogue resistance or a combination of mutations.
• a summary of the genetic methods used to develop lysine producers is given by 0. Tosaka and K. Takinami (Prog. Ind. Microbiol. (Biotechnol. Amino Acids) 24, (1986), 152-172).
• the search for strains that produce amino acids is called screening.
• screening random mutations are induced in a parent strain using common chemical or physical mutagens (e.g. MNNG or UV) and mutants are selected using conventional microbiological methods. Since not all of the selected mutants are amino acid producers, a test for the presence of the desired amino acid follows. This test is usually carried out submerged in shake flasks (10 ml - 2000 ml), whereby the organisms need a sterile nutrient solution (5 - 50 ml) for growth and production, which contains a source of carbon and energy (e.g. glucose, molasses) as well as a nitrogen source (e.g. ammonium sulfate), vitamins (e.g.
• the shake flask test is the decisive grid for the assessment.
• concentration of the amino acid enriched in the medium after 72 to 96 h usually serves as a criterion.
• the production in the shake flask has been completed (K. Nakayama in: Comprehensive Biotechnology 3, 697, 620; ed M. Moo-Young: Pergamon Press (1985)).
• Strains are then selected which secrete the greatest amount of the desired amino acid under the conditions specified in the shake flask.
• This screening method is generally used because it enables a relatively quick selection from the mutants available in each case and is simple in terms of process technology.
• strains selected according to the known screening method which showed the same or comparable conversions, differ significantly when used in a fed-batch process and lead to substantially different amino acid concentrations in the culture medium.
• the invention relates to a process for the selection of productive strains of microorganisms which secrete amino acids in a culture medium inoculated with the microorganisms in question, for use in fed-batch fermentation, characterized in that the concentration of the desired amino acid is determined between the start and end point of the production phase determined at least once and selects the mutant that has excreted the largest amount of this amino acid by this point (s).
• the time generally selected is half of the fermentation period.
• the superiority of the method according to the invention is demonstrated by way of example using L-lysine-producing microorganisms of the genus Corynebacterium glutamicum.
• the term fed-batch process corresponds to a common terminology and denotes a fermentation process in which nutrients such as z. B. carbon sources and nitrogen sources are replenished.
• strains other than those selected according to the usual shake flask test are selected in the process according to the invention.
• the L-Lys concentration was determined after 24 h of shaking incubation, and to determine the final L-Lys concentration after completion of the production after 48 h.
• the examples relate to strains of Corynebacterium glutamicum.
• Strain DM 180-1 is inoculated from the stem tube in 4 ml Caco broth, incubated for 16 h at 30 ° C with shaking and then after 1: 3 dilution in fresh caso broth (4 h, 30 ° C; shaking) in one 100 ml Erlenmeyer flask with chicane containing the test medium (9 ml) inoculated (1 ml).
• the test medium contains 240 g / l molasses, 100 ml / l soy flour hydrolyzate, 12 g / l ammonium sulfate and 10 g / l calcium carbonate. After shaking for 24 h (300 rpm) at 30 ° C, the medium contains 19.0 g / l L-Lys x HCl, after 48 h 32.2 g / l L-Lys x HCl.
• Strain DM 290-2 is grown as described in Example 1 and incubated in a shake flask. After 24 h the medium contains 18.1 g / l L-Lys x HCl, after 48 h 34.4 g / l L-Lys x HCl.
• Strain DM 282-2 is grown as described in Example 1 and incubated in a shake flask. After 24 h the medium contains 24.9 g / l L-Lys x HCl, after 48 h 30.1 g / l L-Lys x HCl.
• the strains with the highest final concentration ie DM 290-2
• DM 282-2 would be selected as having the highest L-lysine concentration after 24 hours.
• Examples 4-6 show how the strains behave in fed-batch fermentation.
• Example 4 Fed batch fermentation DM 180-1
• 0.1 l Caso broth is inoculated with chicane in a 2 l Erlenmeyer flask and incubated with shaking (30 ° C, 24 h, 150 rpm).
• This cell suspension becomes a pre-fermenter with 8 medium M1 (60 g / l molasses, 10 g / l ammonium sulfate, 120 ml / l soy flour hydrolyzate with 1 ml / l phosphoric acid) and after 9 h incubation (33 (C) with stirring and aeration, 1.3 l cell suspension are transferred to the main fermenter (13 l medium M1).
• Example 5 Fed batch fermentation DM 290-2
• Caso broth is inoculated with chicane in a 2 l Erlenmeyer flask and incubated with shaking (30 °, 13 h, 150 rpm).
• a pre-fermenter (10 l medium caso broth with 7.5 g / l sucrose) is inoculated with this cell suspension and incubated for 12 h with stirring and aeration (30 0C).
• the medium contains 37.5 g / lL-Lys x HCl.
• Example 6 Fed batch fermentation DM 282-2
• Caso broth is inoculated with chicane in a 2 l Erlenmeyer flask and shaken incubated (30 ° C, 10 h, 150 rpm).
• This cell suspension is used to inoculate a pre-fermenter (8l medium M1 (60 g / l molasses, 10 g / l ammonium sulfate, 120 ml / l soy flour hydrolyzate with 1 ml / l phosphoric acid) and after 9 hours of incubation (330 ° C) with stirring and aeration 21 cell suspensions are transferred to the main fermenter (10 l of medium M1).
• the medium contains 76 g / l L-Lys x HCl.
• strain DM 282-2 selected according to the production speed criterion according to the invention, is superior in the fed-batch process to that selected according to the conventional final concentration criterion.

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• 1988
  • 1988-07-22 DE DE3824937A patent/DE3824937A1/de active Granted
• 1989
  • 1989-04-22 EP EP89107322A patent/EP0351502B1/de not_active Expired - Lifetime
  • 1989-04-22 DE DE89107322T patent/DE58906674D1/de not_active Expired - Lifetime
  • 1989-07-21 JP JP1187607A patent/JPH0276599A/ja active Pending

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